Retrovirus vectors derived from avian sarcoma leukosis viruses permitting transfer of genes into mammalian cells

ABSTRACT

Recombinant avian sarcoma leukosis virus (ASLV)-derived retrovirus vectors having an expanded host range are described. The host range is expanded by the replacement of the ASLV envelope gene by an envelope gene from a virus capable of infecting both mammalian and avian cells. The resulting recombinant ASLV-derived retroviral vectors can replicate efficiently in avian cells, infect both avian and mammalian cells in high titer, and are replication-defective in mammalian cells. Thus, they are quite safe and advantageous for use in gene therapy and vaccines.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/445,462, filed May 22, 1995, now abandoned.

FIELD OF INVENTION

This invention relates to the fields of genetic engineering and genetransfer. More specifically, the invention relates to recombinantretrovirus vectors derived from avian sarcoma leukosis viruses (ASLVs)having an expanded host range. In particular, this invention relates toASLV recombinant retrovirus vectors wherein a viral env gene derivedfrom a virus capable of infecting both mammalian and avian cells issubstituted for the ASLV env gene, allowing the vectors to efficientlyinfect a wide range of host cells, including mammalian and particularlyhuman cells, in high titers. Additionally, this invention encompassestherapeutic applications employing these vectors.

BACKGROUND OF INVENTION

Retroviral vectors carrying and expressing nucleic acid sequences ofinterest are powerful tools for the transfer of genes into a broad rangeof mammalian cells and into animals, including humans. Indeed,retroviruses offer substantial advantages for use as vectors carryingand expressing desired nucleic acid sequences in both cultured cells andintact animals. (Weiss et al, RNA Tumor Viruses (1982)).

First, the retrovirus life cycle lends itself to the efficient transferof genes into host cells. The infectious retroviral agent is called aviral particle or a virion. Virions consist of a capsid containing theviral genome and any inserted nucleic acid sequences and an envelopemade up of glycoproteins. The envelope glycoproteins on the surface ofthe virion recognize receptors on the host cell that mediate entry ofthe RNA retroviral genome into the host cell. Once inside the host cell,a double stranded DNA copy of the virion RNA genome and any insertednucleic acid sequences of interest is made by a viral enzyme, reversetranscriptase. This DNA copy integrates into the host genome at aprecise point on the viral DNA molecule and at random, or nearly randomsites on host chromosonal DNA. The integrated viral DNA copy is called aprovirus. Since a DNA copy of the viral genome integrates into the hostgenome, the progeny of a single infected host cell are all infected, andthe provirus is located in the same place in the genome of each of theprogeny cells.

Second, in completing their replicative process, retroviruses usually donot lyse the host cell. Thus, the retroviruses constitute an efficientmechanism for the introduction and high level expression of genes inliving host cells.

Third, retroviral genomes are small, making it relatively easy tomanipulate a cloned DNA copy of the genome. Moreover, the viruses areefficient; in culture, essentially all of the cells can be infected.

The ability of the retroviral replication machinery to introduce geneticinformation into the genome of the target cell provided the inspirationfor the development of recombinant retrovirus vectors containing anucleic acid sequence of interest as a vehicle for the stable transferof genes. Moreover, recombinant retroviral vectors have been used in anumber of applications in addition to the expression of genes ofinterest, including insertional mutagenesis, cell lineage studies andthe creation of transgenic animals.

A desirable property useful for the retroviral vector is the ability toreplicate in certain easily manipulated host cells, (e.g., avian cells)allowing rapid replication in these cells without aid of a helper orpackaging cell line. This permits generation of high titer virus stocksby simply passaging transfected cells and allowing the virus to spread.

Another useful property for a retroviral vector is the ability to infecta wide range of host cells, including mammalian, and particularly human,cells in high titers. Preferably, the retroviral vector is unable toreplicate in mammalian cells. Thus, once the vector enters the mammalianhost cell, it becomes a stable provirus, integrated in the host cellgenome and incapable of further rounds of infection in either thepresent or subsequent generations.

A number of retroviral vector systems have been described, includingsystems based on both mammalian (murine leukemia virus, Cepko, et al.,(1984) Cell 37:1053-1062, Cone and Mulligan, (1984) PNAS (U.S.A.)81:6349-6353; mouse mammary tumor virus, Salmons et al., (1984) Biochem.Biophys. Res. Commun. 159:1191-1198; gibbon ape leukemia virus, Milleret al. (1991) J. Virology, 65:2220-2224; human immunodeficiency virus,Buchschacher and Panganiban, (1992) J. Virology 66:2731-2739, Page etal., (1990) J. Virology 64:5270-5276) Shimada et al., (1991) J. Clin.Invest. 88:1043-1047); and avian retroviruses (Boerkoel et al., (1993)Virology 195:669-679, Cosset et al., (1990) J. Virology 64:1070-1078,Greenhouse et al., (1988) J. Virology 62:4809-4812, Hughes et al.,(1986) Poult. Sci. 65:1459-1467, Petropoulos and Hughes, (1991) J.Virology 65:3728-3737, Valsessia et al., (1992) J. Virology66:5671-5676). However, none of these vector systems combines all of theabove features. Indeed, each of the available retroviral vectors suffersfrom certain disadvantages.

For example, one of the most widely used retroviral vectors is areplication-defective derivative of Moloney murine leukemia virus (MLV).The main advantage of MLV is that it has a wide host range and caninfect mammalian host cells, including human cells. However, the vectorsderived from this virus are replication-defective. MLV vectors containall of the cis-active elements necessary for viral replication, but lackthe genes for the viral structural proteins. These proteins must beprovided in trans by a helper or packaging cell line.

MLV and other replication-defective vectors have two majordisadvantages. First, the titers of recombinant retrovirus produced by ahelper or packaging cell line are not always sufficient for someapplications, for example, for in vivo gene transfer experiments or genetherapy. (See, e.g. Hopkins, (1993) PNAS (U.S.A.) 90:8759-8760). Second,recombination events between the helper or packaging cell line genomeand the replication-defective vector can occur and can result in thegeneration of wild-type virus. (Ott et al., (1994) Hum. Gene Ther.5:567-575). Contamination of the recombinant retroviral vector stockwith replication-competent MLV can interfere with gene transfer andpresent potentially serious problems if the vector is used for genetherapy. For example, leukemias and lymphomas were induced in primatesinfected by the wild-type MLV contaminating retroviral vector stocks.(Donahue et al., (1992) J. Exp. Med. 176:1125-1135; Vanin et al., (1994)J. Virology 68:4241-4250). Finally, in order to use a helper orpackaging cell line, a selectable marker must be introduced into theretroviral vector. However, with a helper-independent system there is noneed to introduce a selectable marker into the vector, since anysequence present in the vector will be carried along passively duringreplicants.

Other frequently used retroviral vectors are derived from avian sarcomaleukosis viruses (ASLVs), particularly the Rous sarcoma virus (RSV).(Hughes and Kosik (1984) Virology 136:89-99; Hughes et al., (1987) J.Virology 61:3004-3012). RSV is the only known replication-competentretrovirus carrying an additional gene, oncogene v-src, which isdispensable for viral replication. This oncogene can be deleted from theRSV derived vector and replaced with a gene or genes of interest withoutaffecting the ability of the virus to replicate. For example, retroviralvectors derived from RSV in which the v-src sequences present in theparental RSV have been replaced with a unique restriction site, Cla I,which can be used to insert the gene or genes of interest havepreviously been described. These vectors are designated the RCAS series.(Hughes et al., (1987) J. Virology 61:3004-3102). The stability of thesevectors was improved by removal of the direct repeat upstream of the srcregion. (Hughes et al., (1987) J. Virology 61:3004-3102). Theconstruction and advantages of these vectors are described inPetropoulos and Hughes (1991) J. Virology 65:3728-3737. (See also Hughesand Kosik (1984) Virology 136:89-99). Retrovirus vectors derived fromreplication competent endogenous Rous associated virus type-O (RAV-O)are designated RCOS (Greenhouse, et al., (1988) J. Virology62:4809-4812). Vectors without splice acceptors are designated RCON andRCAN. (Hughes et al., U.S. Pat. No. 4,997,763 (filed Jul. 31, 1987,issued Mar. 5, 1991), Hughes et al., (1987) J. Virology 61:3004-3012,Petropoulos and Hughes, (1991). J. Virology 65:3728-3737, Greenhouse, etal., (1988) J. Virology 62:4809-4812).

In contrast to the replication-defective vectors, recombinant retrovirusvectors based on RSV or other replication competent ASLVs do not requirea packaging or helper cell line. Thus, these vectors can replicate inavian cells without the assistance of helper or packaging cell lines.Consequently, high-titer viral stocks may be easily prepared bytransfecting a plasmid containing the vector into cultured chickenembryo fibroblasts (CEFS) or other avian cells, and passaging thetransfected cells and allowing the virus to spread. The simplicity ofthe virus stock preparation and the high titers that are easilyachievable with the replication-competent retroviral vectors aresignificant advantages. Additionally, these vectors have the desirableproperty of being unable to replicate in mammalian cells. (Federspiel etal., (1994) PNAS (U.S.A.) 91:11241-11245). RSV-derived RCAS vectors havebeen used to express a number of genes and to make transgenic chickens.(Hughes et al., (1990) J. Reprod. Fertil. Suppl. 41:39-49; Petropoulosand Hughes, (1991) J. Virology 65:3728-3737; Petropoulos et al., (1992)J. Virology 66:3391-3397; Salter et al., (1986) Poult. Sci.65:1445-1458; Salter et al., (1987) Virology 157:236-240).

An important limitation of RSV and other ASLV-based vectors is theirhost range. The ASLV-derived vectors disclosed prior to the instantinvention could not efficiently infect mammalian cells. In order toinfect a host cell, the envelope (env) glycoprotein of a retrovirus mustspecifically bind to a cognate receptor on the surface of the host cell.Thus, host range is defined by the binding capability of the envglycoprotein. In RSV and other ASLVs, the env glycoprotein is restrictedto binding to avian cell receptors. Thus, these viruses cannot infectmammalian cells efficiently.

One method that has been used to overcome this limitation is to maketransgenic mice that express the cellular receptor of subgroup A avianleukosis sarcoma viruses. (Federspiel et al., (1994) PNAS (U.S.A.)91:11241-11245). RSV-derived vectors are able to transfer and stablyexpress alkaline phosphatase and chloramphenicol-acetyltransferase (CAT)genes in the muscle of subgroup A receptor transgenic mice. (Federspielet al., (1994) PNAS (U.S.A.) 91:11241-11245). Although the transfer ofgenes is efficient, ASLVs do not replicate in mammalian cells (there isa defect in virion assembly) and the RSV-derived vectors areconstitutively replication-defective in mammalian cells. Moreover, useof the RSV-derived vectors is limited to the small number of mammalianhost cells carrying the subgroup A avian leukosis virus receptor.

Efforts to expand the host range of retroviral vectors to a number ofdifferent cell types in a variety of mammalian species have utilized theability of retroviral capsids to assemble with or be "packaged" by theenvelope glycoproteins of other viral species. Through a mechanism thatis not well understood, a pseudotyped virus bearing envelopeglycoprotein that is a mixture of the two viruses is generated. (Emi etal. (1991) J. Virology 65:1201-1207). The pseudotyped virus has the hostrange of the virus donating the envelope protein. (Burns et al. (1993)PNAS (U.S.A.) 90:8033-8037).

For example, Emi et al. (1991) J. Virology 65:1201-1207 and Burns et al.(1993) PNAS (U.S.A.) 90:8033-8037 describe the generation of pseudotypedviruses by co-infection of the same cells with MLV and the vesicularstomatitis virus (VSV) helper/packaging virus. The resulting pseudotypedviruses have the increased host cell range of VSV (i.e. they can infecthamster cells, which MLV generally cannot infect) but at a low titer).

Landau and Littman, (1992), J. Virology, 66:5110-5113, describe theproduction of replication-defective pseudotyped viruses wherein the MLVgenome bears either the MLV or ecotropic or the RSV envelopeglycoprotein. The packaging system is produced by transient expressionof the env genes in cells infected with replication defective MLV. Theresulting MLV pseudotyped viruses have expanded host ranges.

Miller et al., U.S. Pat. No. 4,861,719 (filed Apr. 25, 1986; issued Aug.29, 1992) and Temin et al., U.S. Pat. No. 5,124,263 (filed Jan. 12,1989; issued Jun. 23, 1992) describe packaging/helper cell lines used toalter the host range of replication defective retroviral vectors theyare co-cultivated with. Both references describe, inter alia,helper/packaging cell lines derived from amphotropic MLV.

Researchers have attempted to "package" the ASLV genome in the envelopeglycoprotein of a virus with a broader host range. For example, Weiss etal., (1977) Virology 77:808-825, describe superinfection of cellsproducing RSV with temperature sensitive mutants of VSV in an effort toexpand the host range of the RSV-based vectors. Two types of pseudotypedviruses resulted: VSV genomes bearing RSV envelope antigens and RSVgenomes bearing VSV envelope antigens. The RSV genomes bearing the VSVenvelope antigens possessed the host range of the VSV virus and werecapable of infecting mammalian cells, but at a lower titer than chickencells.

Weiss and Wong, (1977) Virology 76:826-834 describe mixed infection ofcultured avian cells by RSV and MLV. The appearance of RSV particlesinfectious for mammalian cells was observed. However, the MLV envprotein does not appear to compete efficiently with RSV env to formpseudotyped virus. In addition, the resulting RSV pseudotyped viruseswith the xenotropic and ecotropic MLV env antigens were shown to infectmammalian cells only at a very low titer (on the order of 10² /ml).

In each of these references the expanded host range avian pseudotypedviruses depend on the production of an envelope protein by a help virusor packaging cell. Thus, these vector systems are susceptible torecombination between the two viral genomes and the instability andpotential contamination with wild-type virus recombination engenders.Consequently, they are not suitable for gene therapy.

Some researchers have attempted to expand the host cell range of avianleukosis virus-based vectors by creating recombinant vectors whichexpress chimeric proteins with expanded host cell binding capacity. Forexample, Dong et al., (1992) J. Virology 66:7374-7382, describe arecombinant RSV-based vector expressing a chimeric influenza virushemagglutinin (HA). Plasmids containing chimeric HA genes comprised ofthe coding sequence for the RSV env signal peptide fused to thehemagglutinin (HA) structural genes or a combination of HA and RSVstructural genes were used to co-infect cells with plasmids carrying RSVgag-pol-sequences. Viral particles that contained HA were formed andcould be used to infect mammalian cells. However, thereplication-competence of the vector in avian cells was not demonstratedand the efficiency of infection of mammalian cells was low, on the orderof 10² /ml.

Valsessia-Wittman et al., (1994) J. Virology 68:4609-4619, describe thereplacement of the putative receptor-binding domain of the subgroup (A)RSV envelope protein gp85 (SU) with the peptide known to be the targetfor cellular integrin receptor. Viral particles coated with the modifiedenvelope were shown to infect mammalian cells which are resistant toinfection by subgroup (A) ASLVs. However, infectivity of this vector inmammalian cells also appeared to be quite low, on the order of 10-10²/ml.

In spite of numerous attempts in the prior art to develop vectors thatwere simultaneously able to: 1) efficiently infect a broad range of celltypes in a variety of avian and mammalian species, independent of helperor packaging virus cell lines; and 2) unable to replicate once insidethe mammalian host cell, until the present invention, such a vectorremained elusive.

SUMMARY OF INVENTION

The present invention, in general, provides recombinant avian sarcomaleukosis virus (ASLV)-derived retroviral vectors having an expanded hostrange, thereby allowing the vector to be used for gene transfer in avariety of species. In particular, the ASLV envelope gene is replaced bythe env region derived from a virus capable of infecting both mammalianand avian cells. The recombinant retroviral vectors of this inventioncan replicate efficiently in avian cells, allowing for production ofhigh titer stock and they are able to infect both mammalian and aviancells at a high titer. However, the ASLV-derived vectors of thisinvention are replication defective in mammalian cells, therebyproviding retroviral vectors that are quite safe for use in genetherapy.

It is a general object of the present invention to provide recombinantretroviral vectors that are capable of carrying one or more nucleic acidsequences of interest and expressing them in a broad range of mammaliancells.

It is a further object of the present invention to provide host cells,particularly avian or mammalian cells, carrying the recombinantretroviral vectors and capable of expressing one or more nucleic acidsequences of interest.

It is also an object of the present invention to provide methods forusing the recombinant retroviral vectors to transfer nucleic acidsequences of interest into a broad range of host cell types,, both invitro and in vivo, in a variety of species, including avian andparticularly mammalian species.

It is yet another object of this invention to provide a method for usingthe recombinant retroviral vectors to produce transgenic animals.

It is a further object of the invention to provide transgenic animalscarrying the recombinant retroviral vectors in at least one cell.

It is a further object of the invention to provide methods for treatingor preventing diseases involving transfer by the recombinant retroviralvectors of all or part of a nucleic acid sequence of interest to amammal, preferably a human.

DESCRIPTION OF THE FIGURES

FIG. 1A: A schematic depiction of the construction of plasmid RSVgagpol.

FIG. 1B: A schematic depiction of the construction of recombinantretroviral vector RCAS-M(4070A).

FIG. 2A: The structure of plasmids RSVgagpol and MLVenv shownschematically. RSVgagpol contains the gag-pol open reading frame derivedfrom the retroviral vector RCASBP(A) Petropoulos and Hughes, (1991) J.Virology 65:3728-3737. MLVenv carries a region encoding the signalpeptide, surface glycoprotein gp70 and transmembrane protein p15E of anamphotropic MLV, clone 4070A (Ott et al., (1990) J. Virology 64:757-766,Ott et al., (1992) J. Virology 66:4632-4638).

FIG. 2B: Immunoblotting analysis of the viral particles harvested fromthe complementation assay with RSVgagpol and MLVenv. Antibodies againstRSV p19 (MA) and MLV gp70 (SV) were used. Lanes 1 and 2 show MLVenvrecovered from supernatants of cells transfected with MLVenv plasmidalone. Lanes 3 and 4 show RSVgagpol recovered from supernatants of cellstransfected with RSVgagpol plasmid alone and Lanes 5 and 6 show virusparticles recovered from supernatants of cells co-transfected with bothplasmids.

FIG. 3A: Schematic depictions of the structures of retroviral vectorsRCAS-M(4070A) and RCASBP(A). RCAS-M(4070A) contains a chimeric env genederived from an amphotropic MLV in place of the env gene of RCASBP(A).Retroviral vector RCAS BP(A) was modified to create RCAS-M(4070A) byreplacing the env gene stop codon with a unique NotI cleavage site.Additionally, RSV env coding sequences were removed and the chimericenvelope coding region, including a new stop codon, was insertedgenerating the recombinant retroviral vector RCAS-M(4070A).

FIG. 3B: SDS-PAGE gel. Lanes 1 and 2 contain viral particles recoveredfrom the transfection of CEFs with RCAS-M(4070A). Lanes 3 and 4 arecontrols and contain the viral particles recovered from CEFs infectedwith pR4070A, the molecular clone of the amphotropic MLV from which theMLV env gene was derived.

FIG. 4A: Stained cells expressing gp70 from RCAS-M(4070A). To measurethe ability of RCAS-M(4070A) to replicate in CEFs, cells weretransfected with RCAS-M(4070A) DNA and passaged six times to allow thevirus to spread. At passages 1, 4 and 6 a small number of cells wereplated separately. Those containing virus were identified by stainingwith antibodies that react with gp70. Only a small number of positivelystained cells are seen in passage 1. This number increases significantlyby passage 4. At passage 6 virtually all cells are infected andexpressing gp70.

FIG. 4B: Immunoblot analysis of the viral particles recovered from theharvested CEFs cell medium at each passage with antibodies against p19and gp70. The amount of viral proteins in the supernatant increases bypassage 4 and reaches a high level by passage 6.

FIG. 5: Puromycin-resistant colonies of D17 (dog) cells produced byinfecting D17 cells with serial dilutions of RCAS-M(4070A)Puro.

FIG. 6A: Schematic Structure of RCAS-M(4070A)NEO. Restrictionendonuclease sites are indicated. "E" refers to EcoRI, "B" refers toBamHI.

FIG. 6B: Detection of RCAS-M(4070A)NEO provirus in the genomic DNA ofinfected NIH 3T3 cells by Southern blot hybridization. Lanes 1-19 showfragments derived by cleavage of the DNA of G418^(r) clones by EcoRI andBamHI. Lane C shows fragments derived by cleavage of plasmidRCAS-M(4070A) DNA by EcoRI and BamHI.

FIG. 6C: Detection of RCAS-M(0470A)NEO provirus in the genomic DNA ofinfected HeLa cells by Southern blot hybridization. Lanes 1-15 showfragments derived by cleavage of the DNA of G418^(r) clones by EcoRI andBamHI. Lane C shows fragments derived by cleavage of plasmidRCAS-M(4070A) DNA by EcoRI and BamHI.

FIG. 7: Immunoblot analysis of viral particles recovered from theculture of cells infected with RCAS-M(4070A). Antibodies against p15Ewere used. Lane 1 shows wild type MLV p15E proteins Lane 2 shows p15Eproteins from a strain of MLV with a defective protease gene. Lane 3shows RCAS-M(4070A) p15E proteins.

FIG. 8: A schematic depiction of mutations in the amphotropic env geneof RCAS-M(4070A)NEO. CEFs were infected with RCAS-M(4070A)NEO after 3passages of the virus. Full-length clones of the viral DNA were derivedfrom the library of a low-molecular-weight DNA which was extracted frominfected CEFs. Env genes of clones env 1, 3, 6, 8 and 9 were sequencedand are depicted.

FIG. 9A: Replication of RCAS-M2(4070A)Puro and RCAS-M2(4070A)Puro 1, 6,8, and 9 in CEFs as measured by a RT assay. CEFs were transfected withplasmid DNA and passaged. 24 h after transfection and at each passage,virus particles were recovered from the culture fluid by centrifugationand quantified by determination of the RT activity. The control,uninfected cells, is designated a "mock." RCAS-M2(4070A)Puro isdesignated "env (wt)" and RCAS-M2(4070A)Puro 1, 6, 8 and 9 aredesignated "env 1", "env 6", "env 8", and "env 9", respectively.

FIG. 9B: Titers of RCAS-M2(4070A)Puro1, 6, 8 and 9 on D17 cells. Cellswere infected with the serial 10-fold dilutions of virus-containingculture fluid harvested at each passage. Resistant clones were selectedin the medium containing puromycin. pac^(r) colonies were stained withGiemsa stain and counted. RCAS-M2(4070A)Puro 1, 6, 8 and 9 aredesignated "env 1", "env 6", "env 8", and "env 9", respectively. "Cfu"means colony-forming units.

FIG. 9C: Titers of RCAS-M2(4070A)Puro8 (designated "RCAS-M2(4070A)Puro")and RCAS-M2C(4070A)Puro on D17 cells. Values shown are the average oftwo independent determinations.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to recombinant retroviral vectors capable ofinfecting a broad range of host cells, in particular both mammalian andavian cells, in high titer. In general, the recombinant retroviralvectors of this invention comprise at least one long terminal repeat(LTR), a gag region, a pol region, and an env region, or functionalequivalents thereof. More specifically, the recombinant retroviralvectors comprise LTR, pol and gag regions derived from avian sarcomaleukosis viruses (ASLV) and an env region derived from a virus capableof infecting both avian and mammalian cells, such as amphotropic MLV.The recombinant vectors are capable of carrying and expressing one ormore nucleic acid sequences of interest, replicate efficiently in aviancells and are able to efficiently infect and transfer genes into a broadrange of mammalian cells. The recombinant retroviral vectors of theinvention are replication-defective in mammalian cells, greatlyenhancing their safety.

Gag is the retroviral gene which encodes the structural proteins thatform the virion core. The gag region of the vector may be derived fromthe gag region of any member of the ASLV family. Examples of the genomesfrom which the gag sequence may be derived include, but are not limitedto, Rous sarcoma virus (RSV), MC29-associated virus (MAV), Rousassociated virus (RAV), RAV-O, avian erythroblastosis virus (AEV), avianmyoblastosis virus (AMV), other members of this virus family and theirassociated helper viruses. The gag region may comprise all or parts ofthe gag gene. For a replication competent vector, it is preferable thata sequence sufficient to encode a functional gag protein is used. In apreferred embodiment the gag region is derived from RSV.

Pol is the retroviral gene which encodes the reverse transcriptase andintegrase. The pol region may be derived from the pol region of anymember of the ASLV family. Examples of the genomes from which the polsequences may be derived, include, but are not limited to, RSV, MAV,RAV, RAV-O, AEV, AMV, other members of this virus family and theirassociated helper viruses. The pol region may comprise all or parts ofthe pol gene. For a replication competent vector it is preferable that asequence sufficient to encode a functional pol protein is used. By wayof example, a pol region derived from the Bryan high titer RSV strainmay be used. (Petropoulos and Hughes (1991) J. Virology, 65:3728-3737).

Env is the retroviral gene which encodes the envelope antigens thatdetermine the antigenic and subgroup specificity of the virus. The envsequence is preferably derived from the envelope region of a viruscapable of infecting both mammalian and avian cells. Examples of thegenomes from which the env sequences may be derived include, but are notlimited to, mammalian viruses capable of infecting avian species, suchas the amphotropic Moloney murine leukemia virus (MLV) env sequence.Weiss et al., (1982) RNA Tumor Viruses; Weiss (1985) Supplement to RNATumor Viruses. The env sequence may comprise all or parts of the envgene. For a replication competent virus, it is preferable that asequence sufficient to encode a functional envelope protein is used. Byway of example, a sequence encoding the complete amphotropic MLV envregion may be used in the recombinant retroviral vector of thisinvention.

In a preferred embodiment, the coding sequence of a virus capable ofinfecting both mammalian and avian cells such as amphotropic MLV, is"adapted" to permit initial replication rates similar to those observedin wild type RSV via one or more mutations of the DNA of the env codingsequence, including, but not limited to, the substitution of the prolineat position 242 with isoleucine. As described more fully in theexamples, the "adapted" env sequence may be produced by passaging thevirus and selecting for fast replicating clones. Additionally, oneskilled in the art will recognize that the env sequence may be "adapted"by a number of other methods including, but not limited to, use of sitedirected mutagenesis to produce the desired mutation(s).

Alternatively, a chimeric envelope sequence may be used in constructingthe recombinant retroviral vector of this invention, comprising thecoding region of a wild type or "adapted" env gene sequence of a viruscapable of infecting mammalian and avian cells and the N terminal signalpeptide sequence of an ASLV.

Long terminal repeats (LTRs) facilitate integration of the viral genomeinto the host genome and contain promoters for transcription of theviral genome. The retroviral vectors of this invention have at leastone, preferably two LTR's. A LTR derived from an ASLV family member maybe used. These include, but are not limited to, RSV, MAV, RAV, RAV-O,AEV, AMV and other members of this family, and their associated helpervirus. (Hughes and Kosik, (1984) Virology 136:89-99, Hughes et al.,(1987) J. Virology 61:3005-3102). For a replication competent virus,preferably the entire LTR is used. Alternatively, in another embodiment,a retroviral vector with a single LTR may be used. If a single LTRcircular plasmid is used, a two LTR proviral form can be simply derivedby cleavage at an appropriate restriction site and ligation. (Hughes andKosik, (1984) Virology 136:89-99).

The RSV-derived vector designated RCAS may be used to generate therecombinant retroviral vector of this invention by providing the LTR,pol and gag sequences. (Hughes et al., J. Virology (1987) 61:3004-3012;Hughes and Kosik (1984) Virology 136:99-99, herein incorporated byreference). Additionally, in another embodiment the RCASBP vectors areused. (Petropoulos and Hughes, (1991) J. Virology 65:3728-3737). In theconstruction of RCAS, the genome of a circular DNA form of the SchmidtRappin A (SR-A) strain of RSV, was recloned in proviral DNA form withthe E coli replicon between the LTRs of the provirus. The v-src codingregion was removed and was replaced with the cleavage site for therestriction endonuclease Cla I. One copy of the direct repeats flankingv-src in SR-A was removed. To construct RCASBP, the RCAS vector was usedand the pol gene of the SR-A strain was replaced by the pol gene of theBryan high titer strain of RSV.

Conventional methodology may be used to replace the RSV-derived envsequence present in the RCAS or RCASBP vector with the env sequence ofthe amphotropic MLV retrovirus (see Example 2). The complete MLV envcoding sequence may be used or a chimeric env sequence comprising theMLV env coding sequence having the RSV signal peptide in place of theMLV signal peptide may be constructed and cloned into the vector. Theresulting retrovirus, designated RCAS-M (4070A), is shown in FIG. 3. Thefunctional equivalents of the recombinant retrovirus shown in FIG. 3 arealso intended to be encompassed by this invention.

In a preferred embodiment, the of RCAS-M (4070A) is "adapted" for fastinitial replication by introduction of one or more mutations into theenv sequence, including substitution of the proline at position 242 withan isoleucine. The resulting retrovirus is designated RCAS-M2(4070A).The functional equivalents of the RCAS-M2 (4070A) recombinant retrovirusare also intended to be encompassed by this invention.

In yet another embodiment of this invention, the envelope region of therecombinant retroviral vectors of the invention is derived from a virusthat recognizes mammalian, but not avian cell receptors. In order for avector containing such an envelope sequence to infect avian host cells,the avian cells may be engineered to express a receptor recognized bythe chosen envelope region.

The recombinant retroviral vectors of this invention may be used forexpression of nucleic acid sequences of interest. In this embodiment,the vector further comprises one or more nucleic acid sequences encodinga protein of interest. One skilled in the art will recognize thatvirtually any nucleotide sequence from virtually any genome may be used.In particular, nucleic acid sequences encoding a therapeutic orimmunogenic protein may be used. Alternatively, nucleic acid sequencesencoding the antisense strand for one or more proteins may be used toinhibit the expression of the proteins. The nucleic acid sequences maybe inserted into the retroviral vector of this invention usingconventional methodology known to those skilled in the art. Examplesinclude, but are not limited to, cleavage by a specific restrictionendonuclease and ligation or homologous recombination.

Conventional methodology including, but not limited to site directedmutagenesis, may be used to introduce one or more restrictionendonuclease sites into the vectors of this invention. (Sambrook et al(eds) (1989) "Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y. and Ausubel et al. (eds) (1987) in "CurrentProtocols in Molecular Biology" John Wiley and Sons, New York, N.Y.). Toretain the ability of the vectors to replicate in avian cells it isimportant not to delete any of the gag, pol, or env sequences necessaryfor virus replication. Any restriction endonuclease site may begenerated, preferably an endonuclease unique to the retroviralexpression vector or an endonuclease site that does not appearfrequently in the vector. One of skill in the art will know how toselect and create the appropriate restriction endonuclease sites.

In one embodiment, RSV is used to generate the recombinant retroviralvectors of the invention and the nucleic acid sequences of interest maybe inserted in place of the RSV v-src oncogene using restrictionendonucleases and ligation. In a preferred embodiment, the nucleic acidsequences of interest are inserted into the Cla I site of theRSV-derived RCAS or RCASBP vectors and at least one copy of the directrepeats flanking the v-src site are removed to increase the stability ofthe vector (see Example 2).

The desired nucleic acid sequences inserted into the recombinantexpression vector of this invention may be transcribed under thedirection of the LTR promoter. By way of example, the RSV-derived RCASor RCASBP vectors may be used to generate the recombinant retroviralvectors of the invention when the LTR will direct the expression of thenucleic acid sequence of interest. (Hughes and Kosik, (1984) Virology136:89-99, Hughes et al., (1987) J. Virology 61:3004-3012).

Alternatively, the expression of the desired nucleic acid sequences maybe under the direction of an internal promoter within the recombinantretroviral vector of this invention. By way of example, tissue specificpromoters such as a sk actin (Petropoulos and Hughes (1991) J. Virology65:3728-3737), inducible promoters such as metallothionien (Petropoulosand Hughes (1991) J. Virology 65:3728-3737), a cell cycle or stagespecific promoters may be used to direct expression of the desirednucleic acid sequences. One of skill in the art will know what promotershould be used based on the intended application of the gene product. Byway of example, the RSV-derived RCAN vectors, (Hughes and Kosik, (1984)Virology 136:89-99; Hughes et al. (1987) J. Virology 6:3004-3012) may beused to generate the recombinant retroviral vectors of the inventionwhen an internal promoter will be used. Conventional methodology may beused to incorporate these promoters into the recombinant retroviralvectors.

Additionally, one skilled in the art will recognize that a spliceacceptor or an IRES (internal ribosome entry site) may be introduced inthe retroviral vector to permit expression of an inserted gene.

The recombinant vectors of this invention may further include additionalexpression control elements, including, but not limited to enhancersequences. Conventional methodology can be used to incorporate theseadditional expression control elements into the vector. (Sambrook et al(eds (1989) "Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y. and in Ausubel et al. (eds) (1987) in "CurrentProtocols in Molecular Biology" John Wiley and Sons, New York, N.Y.).

Another aspect of the present invention is to provide a method for usingthe recombinant retroviral vectors to transfer one or more nucleic acidsequences of interest to a broad range of host cells from a variety ofspecies, particularly mammalian species. The recombinant retroviralvectors of the present invention may carry one or more nucleic acidsequences of interest. The virions encapsulating the nucleic acidsequences of the invention are capable of infecting a broad range ofmammalian cell types. Once inside the cell, the recombinant retroviralvectors make a DNA copy of the retroviral genome and the nucleic acidsequences of interest, which is stably inserted into the host genome andcan be expressed by the host cell.

The recombinant retroviral vectors of the invention may be directlyintroduced into a host cell or may be inserted into a plasmid or otherconstruct which is then inserted into a host cell. For example, togenerate large quantities of the retroviral vector DNA of thisinvention, the recombinant retroviral vectors of the invention may becloned into a plasmid for propagation of the DNA in appropriate cells.Examples of the cells that may be used include but are not limited to E.coli. Construction of the plasmid containing the retroviral vector DNAcan be performed by conventional methods.

By way of example the pBR322 plasmid or a derivative thereof may be usedto propagate the vector DNA. In a preferred embodiment the retroviralvector of the invention is inserted into the E. coli replicon pPH,derived from pBR322. (Hughes et al. (1987) J. Virology 61:3004-3012).

It will be understood by one skilled in the art that the plasmidcarrying the recombinant retroviral DNA should contain additionalelements necessary for transfer and subsequent replication of theconstruct in the host system being used to propagate the DNA. Examplesof such elements include but are not limited to origins of replicationand selectively. It will be understood by one skilled in the art thatthe correct combination required or preferred elements to be used.

The means by which the recombinant retroviral vectors may be introducedinto the host cell include, but are not limited to, microinjection,electroporation, transduction or transfection using DEAE-dextran,lipofection, calcium phosphate or other procedures known to one skilledin the art (Sambrook et al. (1989) in "Molecular Cloning. A LaboratoryManual," Cold Spring Harbor Press, Plainview, N.Y.).

Another aspect of this invention is to provide host cells into which therecombinant retroviral vector containing all or part of nucleic acidsequences of interest has been inserted. The host cells containing therecombinant retroviral vector of this invention include eukaryotes, suchas avian species including ducks, chickens, turkeys, and quail, andmammalian species including mice, dogs, humans. Preferred host cellsinclude, but are not limited to, CEF cells, NIH 3T3 cells, D17 cells andHeLa cells.

The retroviral vectors of this invention are replication competent inavian cells. Avian cells including but not limited to, chicken, turkeys,quail, and duck may be used to generate a viral stock. The viral stockmay be generated by methods known to those skilled in the art. Therecombinant retroviral vector cloned into a plasmid may be introducedinto the avian cells by conventional methodology and the cells may bepassaged to allow the virus to spread throughout the culture. Subsequentgenerations of the virus may be used to infect other cells.Alternatively, a virus carrying the recombinant retroviral vector may beused to infect the avian cells. The viral stock produced may be used toinfect other host cells. Examples of cells that may be infected includebut are not limited to, duck, chicken, turkeys cells or mammalian cells.Examples of mammalian cells include but is not limited to hematopoieticstem cells, islet cells, or T-cells.

Host cells containing a recombinant retroviral vector of the inventioncan be used in vitro to produce recombinant proteins. The recombinantproteins can be isolated and purified by conventional methods. (Sambrooket al (eds) (1989) "Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Press, Plainview, N.Y. and in Ausubel et al. (eds) (1987) in"Current Protocols in Molecular Biology" John Wiley and Sons, New York,N.Y.). Additionally, host cells may be used in vivo to supplyrecombinant proteins.

In another embodiment, the recombinant retroviral vectors of theinvention may be used to transfer nucleic acid sequences encoding one ormore therapeutic proteins to a mammal, preferably a human, in need ofgene therapy. The retroviral vector carrying the nucleic acid sequenceof interest may be administered to an individual in need of such therapyin a variety of ways. Retroviral supernatants from avian host cellstransfected with and producing the virus may be administered to theindividual in need of gene therapy. Additionally, a substantiallypurified form of the virus may be administered to the mammal in need ofsuch treatment alone or in the form of a pharmaceutical composition.

Alternatively, the gene therapy may be accomplished by inserting thenucleic acid sequences encoding the therapeutic protein(s) into therecombinant retrovirus vector of the invention and introducing it into ahost cell. The host cell which contains the recombinant retroviralvector and expresses the desired therapeutic protein is thenadministered to or implanted in the individual in need of gene therapy.The cells then express the therapeutic protein recombinantly in themammal.

The host cells may be from virtually any species. In one embodiment, thehost cells are taken from the individual in need of the gene therapy.Examples of such host cells include, but are not limited to,hematopoietic stem cells or T cells. In another embodiment the hostcells are not from the individual receiving the therapy, but are fromdifferent species. For example, in one embodiment, which takes advantageof the ability of the retroviral vectors of the invention to replicateand produce virus in avian but not mammalian cells, avian host cellscontaining the retroviral vector are administered to a mammal in need oftherapy.

Means of administering the host cell containing the recombinantretroviral vectors of the invention which recombinantly express theproteins of interest include, but are not limited to, intravenous,intramuscular, intralesional, subcutaneous or intraperitoneal injectionor implantation. Alternatively, the cells containing the recombinantretroviral vectors may be administered locally by topical application,direct injection into an affected area or implantation of a porousdevice containing cells from the host or another species in which therecombinant retroviral vectors are inserted and which express theproteins of interest.

Examples of diseases that may be suitable for gene therapy include, butare not limited to, neurodegenerative diseases or disorders,Alzheimer's, schizophrenia, epilepsy, neoplasms, cancer and AIDS.

In yet another aspect of the invention, the recombinant retroviralvectors can be used to generate transgenic animals carrying therecombinant vector in at least one cell. In a preferred embodiment, therecombinant retroviral vector carrying one or more nucleic acidsequences of interest is introduced into an animal or an ancestor of theanimal at an embryonic stage. The transgenic animal can be made byseveral methods, including, but not limited to, introducing therecombinant retroviral vector carrying the nucleic acid sequences ofinterest into the embryonic animal by infection or injection.Additionally, the recombinant retroviral vector may be inserted intohost cells which are then introduced into the developing embryo. By wayof example, chicken cells containing the recombinant retroviral vectorand carrying the gene of interest can be implanted into the blastocyststage of a mammalian embryo.

Examples of animals into which the recombinant retroviral vector can beintroduced include, but are not limited to, non-human primates, cows,sheep, dogs, mice, rats, or other rodents. Such transgenic animals maybe useful as biological models for the study of disease and to evaluatediagnostic or therapeutic methods for disease.

It will be appreciated by those skilled in the art that the recombinantretroviral vectors of this invention may be used to generate transgenicanimals without additional nucleic acid sequences (e.g. insertionalmutagenesis). Alternatively, nucleic acid sequences of interest may beinserted into the recombinant retroviral vectors of the invention suchthat the transgene animal expresses the desired gene(s).

It is a further aspect of the invention to use the recombinantretroviral vectors to deliver a prophylactic or therapeutic vaccine fora wide variety of mammalian, and particularly human, diseases.

A prophylactic vaccine is provided in advance of any evidence of thedisease of interest and serves to prevent or attenuate the disease in amammal. In a preferred embodiment, mammals, preferably humans, at highrisk for the disease of interest, are treated prophylactically with thevaccines of this invention.

When provided therapeutically, the vaccine is provided at or shortlyafter the onset of the disease of interest (or symptoms of the diseaseof interest) to enhance the immune response of a patient (a mammal,preferably a human) to the disease of interest and to attenuate thedisease.

The vaccine, which acts as an immunogen, may be in the form of a celltransfected with the recombinant retroviral vector of the inventioncarrying nucleic acid sequences encoding all or part of one or moreimmunogenic peptides associated with the disease of interest. Thevaccine may also be in the form of a culture supernatant from cellsproducing viral particles or a preparation of viral particles containingthe retroviral vectors of interest with the inserted nucleic acidsequences of interest.

When the vaccine is used in the form of a host cell, the recombinantretroviral vector of the invention can be introduced into virtually anymammalian cell. The means by which the vector carrying the gene may beintroduced into a cell include, but are not limited to, infection orother procedures known to one skilled in the art (Sambrook et al. (eds)(1989) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y.). The mammalian cells infected with the recombinantretrovirus vectors of the invention and expressing the immunogenicprotein can be administered to mammals and serve as a vaccine. Cellsexpressing the immunogenic protein of interest can be administeredintravenously, intramuscularly, intraperitoneally, intralesionally orsubcutaneously.

Additionally, the vaccine may be administered as a preparation of viralparticles. Viral particles containing the recombinant retroviral vectorsmay be directly administered to the mammal several ways, including, butnot limited to exposure of cells to virus ex vivo or injection of theretrovirus into the affected tissue or intravenously. Alternatively,viral particles carrying all or part of the nucleic acid sequences ofinterest may be administered locally by direct injection into anaffected area or by topical application in a pharmaceutically acceptablecarrier.

Examples of mammals to which the vaccine may be administered include,but are not limited to, mice, rats, dogs, non-human primates, and humanswith a family history of the disease of interest. Veterinary uses areintended to be encompassed by this invention.

Vaccination can be conducted by conventional methods. The recombinantviral vectors containing the nucleic acid sequences encoding theimmunogenic protein may be administered once or at periodic intervalsuntil a significant titer of antibody or immune cells against theimmunogen protein of interest is produced. One skilled in the art willknow the proper immunoassays and other methods for detecting andmeasuring the presence of immune cells against the protein of interest.

The recombinant retroviral vector expressing the immunogen may beadministered in a pure or substantially pure form. Additionally, it ispossible to present it as a pharmaceutical composition, formulation orpreparation.

The pharmaceutical compositions, formulations or preparations of thepresent invention, both for veterinary and for human use, may comprisean immunogen as described above, together with one or morepharmaceutically acceptable carriers and, optionally, other therapeuticingredients. The carrier(s) must be "acceptable" in the sense of beingcompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof. The formulations may convenientlybe presented in unit dosage form and may be prepared by any methodwell-known in the pharmaceutical art.

All methods include the step of bringing into association the activeingredient with the carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product into the desired formulation.

Formulations suitable for intravenous, intramuscular, subcutaneous, orintraperitoneal administration conveniently comprise sterile aqueoussolutions of the active ingredient with solutions which are preferablyisotonic with the blood of the recipient. Such formulations may beconveniently prepared by dissolving solid active ingredient in watercontaining physiologically compatible substances such as sodium chloride(e.g. 0.1-2.0 M), glycine, and the like, and having a buffered pHcompatible with physiological conditions to produce an aqueous solution,and rendering said solution sterile. These may be present in unit ormulti-dose containers, for example, sealed ampoules or vials.

The formulations of the present invention may incorporate a stabilizer.Illustrative stabilizers are polyethylene glycol, proteins, saccharides,amino acids, inorganic acids, and organic acids which may be used eitheron their own or as admixtures. These stabilizers are preferablyincorporated in an amount of 0.11-10,000 parts by weight per part byweight of immunogen. If two or more stabilizers are to be used, theirtotal amount is preferably within the range specified above. Thesestabilizers are used in aqueous solutions at the appropriateconcentration and pH. The specific osmotic pressure of such aqueoussolutions is generally in the range of 0.1-3.0 osmoles, preferably inthe range of 0.8-1.2. The pH of the aqueous solution is adjusted to bewithin the range of 5.0-9.0, preferably within the range of 6-8. Informulating the immunogen of the present invention, anti-adsorptionagent may be used.

After immunization, the efficacy of the vaccine can be assessed byproduction of antibodies or immune cells that recognize the antigen, asassessed by techniques known to those skilled in the art. If the mammalto be immunized is already afflicted with the disease of interest, thevaccine can be administered in conjunction with other therapeutictreatments.

One of skill in the art will know the parameters to determine thecorrect titer of particles to be administered. The quantity ofrecombinant retroviral vector or virus carrying all or part of thenucleic acid sequence encoding the immunogenic protein of interest to beadministered may be based on the titer of virus particles. The amount ofvirus to be administered is in no way limited to a particularconcentration and may vary depending upon the individual being healed.Based on clinical parameters the treating physician will determine thetherapeutically effective amount of the virus containing the gene ofinterest to be administered to a given individual. Such therapy may beadministered as often as necessary and for the period of time judgednecessary by the treating physician. The therapeutic methods describedherein may be used alone or in conjunction with additional therapy knownto those skilled in the art for the treatment of a given disease orcondition in a mammal.

All books, articles, or patents referenced herein are incorporated byreference. The following examples illustrate various aspects of theinvention, but in no way are intended to limit the scope thereof.

EXAMPLE 1 The Amphotropic MLV Env Protein Efficiently Assembles with RSVGag Proteins

In order to investigate directly the ability of RSV gag and MLV env toassemble, a complementation assay in which gag proteins of RSV and theenvelope glycoproteins of MLV were transiently expressed in chickenembryo fibroblasts (CEFs) derived from EV0 chicken embryos wasdeveloped. (Astrin et al., (1979) Nature 282:339-341. See also Hughesand Kosik (1984) Virology 136:89-99, Hughes et al., (1987) J. Virology61:3004-3012). The CEFs used in this experiment and the other exampleswere maintained in Dulbecco modified Eagle medium (DMEM, GIBCO BRL, MD)supplemented with 5% fetal bovine serum, 5% newborn calf serum, 10%tryptose-phosphate broth (GIBCO BRL), 100 u/ml penicillin and 100 μg/mlstreptomycin.

Construction of the Plasmids

Two plasmids, RSVgagpol (which contains the gag-pol open reading framederived from the retroviral vector RCASBP(A) (Petropoulos and Hughes,(1991) J. Virology 69:3728-3737)) and MLVenv (which carries a regionencoding the signal peptide, surface glycoprotein gp70 and transmembraneprotein p15E of an amphotropic MLV (clone 4070A))(FIG. 2A) wereconstructed by standard methods. See Sambrook et al., (eds) (1989)Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y. Recombinant clones were isolated in E. coli DH5a.

Plasmid RSVgagpol was constructed as follows: plasmid DNA RCASBP(A)(Petropoulos and Hughes, (1991) J. Virology 69:3728-3737 was cleaved byrestriction endonucleases Sac I and Xba I (FIG. 1A) and the fragmentcontaining the complete gag region and the 3'-end of pol was isolated.In a separate reaction, RCASBP(A) was digested by Xho I. The Xho I endswere filled in using T4 DNA polymerase and the DNA was subsequentlydigested by Xba I. The fragment Xba I-Xho I(blunt) containing the 3'-endof the pol open reading frame was isolated. The Sac I-Xba I and XbaI-Xho I (blunt) fragments were ligated in the presence of pBluescript KSII DNA that had been cleaved by Sac I and Eco RV. The resulting plasmidpT7gagpol contained the whole gag-pol region flanked by unique Sac I andCla I sites. RCASBP(A) was digested by Sac I and Cla I to removegag-pol-env region, and Sac I-ClaI fragment from pT7gagpol was inserted.

To construct plasmid MLVenv, the env coding region of the amphotropicMLV was PCR-amplified from a plasmid pR4070A (Ott et al., (1990) J.Virology 64:757-766, Ott et al., (1992) J. Virology 66:4632-4638)(kindly provided by Alan Rein, ABL-Basic Research Program) with theforward primer AMPH-F (AAAAGAGCTCGGCCGACACCCAGAGTGGAC) (SEQ ID NO: 1)located just upstream env initiation codon and the reverse primer AMPH-R(AAAAGAGCTCTCATGGCTCGTACTCTATGGGTT) (SEQ ID NO:2)spanning the envtermination codon. The resulting Sac I recognition sites were includedin the 5'-ends of both primers. PCR product was cleaved by Sac I andinserted into the vector TFA-NEO (Federspiel et al., (1989) J. Virology173:167-177), generating MLVenv. This construct expresses amphotropicenv gene under the transcriptional control of the RSV LTR.

Transfection of Cells and Preparation of Virus Particles

Plasmids RSVgagpol and MLVenv were introduced into CEFs by transfectionboth separately and in the mixture. Calcium phosphate-mediatedtransfection of plasmid DNAs into CEFs was performed according tostandard procedures Graham and Van der Eb, (1973) J. Virology52:456-467. Precipitates containing 10 μg of DNA per 10 mm-plate wereincubated with subconfluent CEF monolayers for 6 hr at 37° C. followedby the incubation with the medium containing 15% glycerol for 5 min at37° C. Cells were washed twice with the phosphate-buffered saline (PBS)and incubated in a growth medium for 24 hr. When necessary, transfectedcells were passaged to allow the virus to spread through the culture.

Virus-containing culture fluid was then harvested. 24 hr aftertransfection, the culture medium was harvested and viral particles wererecovered by ultracentrifugation. To prepare virus particles, culturefluid was clarified by low-speed centrifugation and the virus waspelleted through 15% sucrose cushion in SW41 rotor (Beckman) at 35,000rpm for 1 hr at +4° C. The resulting pellet was resuspended in proteinsample buffer and heated at 100° C. for 4 min before loading on a gel.

Immunoblot Analysis

Viral proteins were resolved by electrophoresis in a 4-20% gradientsodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Afterelectrophoresis proteins were electroblotted onto a nitrocellulosemembrane (BA85, Schleicher & Schuell, NH). RSV matrix protein wasdetected by incubation with rabbit antiserum against p19 (MA)(generously provided by Volker Vogt, Cornell University). MLV envelopeglycoproteins were detected with goat antiserum against gp70 (SU).Protein bands were visualized using enhanced chemiluminescence (ECL)detection system (Amersham).

The immunoblotting analysis (FIG. 2B) showed that MLVenv plasmid, whenintroduced into CEFs, expresses surface glycoproteins that are anchoredon a cell membrane and can be detected by immunoblotting in a membraneprotein fraction (data not shown) but are not secreted into the medium(FIG. 2B, lanes 1 and 2). Transfection of RSVgagpol results in asynthesis of capsid proteins that assemble into virus-like particlesthat can be recovered by ultracentrifugation (FIG. 2B, lanes 3 and 4).More than one band was detected by the anti-p19 antibodies. Since RSV MAis phosphorylated in several positions, additional bands usually seen onimmunoblots apparently represent proteins with different degree ofphosphorylation. Burstein et al., (1992) J. Virology 66:1781-1785.

Co-transfection of RSVgagpol and MLVenv causes the co-expression of bothcapsid and envelope proteins in the same cell. Immunoblotting analysisshows that both the RSV gag and MLV env are present in the particlesproduced upon co-transfection (FIG. 2B, lanes 5 and 6) suggesting thatpseudotyped virions are formed.

This data shows that the amphotropic MLV envelope can associateefficiently with an RSV virion if competing RSV envelope is not present.

EXAMPLE 2 The Recombinant Retroviral Vector RCAS-M(4070A) Constructionof RCAS-M(4070A)

Given the apparent efficiency with which the MLV envelope proteinassembles with RSV gag, recombinant RSV genome was constructed in whichthe envelope gene in the parental virus was replaced with the env geneof an amphotropic MLV. To ensure the efficient intracellular transportof the envelope precursor and the signal peptide cleavage in aviancells, a chimeric env gene in which a sequence encoding a mature MLVgp70 was fused to the RSV envelope signal peptide. RSV env codingsequences were removed and the chimeric envelope coding region,including a new stop codon was inserted, generating the recombinantretroviral vector RCAS-M(4070A).

The recombinant retroviral vector RCAS-M(4070A) was prepared as follows.First, an Eco RI fragment of RCASBP(A) spanning the 3'-end of the RSVenv gene and U3R segment of the 3'-LTR was subcloned in pUC19, givingrise to pUCenvRI (FIG. 1B). The termination codon for env was replacedby a Not I site using site-directed mutagenesis. Clones containingmutant plasmids with the Not I site were propagated in Escherichia coliBMH 71-18 mutS (Clontech) and selected by Not I cleavage. The resultingconstruct was called pUCenvRINOT (FIG. 1B).

The env gene of RCASBP(A) was replaced with the one carrying Not I site.RCASBP(A) was digested by Kpn I and Cla I and the fragment containingenv region was removed. The env region containing the unique Not I sitewas introduced by ligating a Kpn I-Eco RI fragment of RCASBP(A) and theEco RI-Cla I fragment from pUCenvRINOT with RCASBP(A) that had beencleaved by Kpn I and Cla I, generating plasmid RCASBP(A)NOT (FIG. 1B).

Overlap extension, Horton et al., (1990) Biotechniques 8:528-535, wasused to construct a chimeric amphotropic env gene in which the sequencecoding for the N-terminal signal peptide of MLV was replaced with theequivalent sequence from the RSV env gene. A fragment spanning theunique Kpn I site, the env splice acceptor site and the signal peptidewas amplified from RCASBP(A) by PCR using primers RSV-FOR(GGACGAGGTTATGCCGCTGTG) (SEQ ID NO: 3) and RSV-BACK(ACATTAAAGACCTGATGGGGGCTAACATCAGCTCTTACCCCCGTAA) (SEQ ID NO: 4) (FIG.1B). The fragment beginning with the codon for the first serine residueof the mature gp70 and spanning the whole env open reading framefollowed by the unique Not I site was amplified from pR4070A with theprimers MLV-FOR (AGCCCCCATCAGGTCTTTAATGT) (SEQ ID NO: 5) and MLV-BACK(AGCGGCCGCTCATGGCTCGTACTCTATGGGTT) (SEQ ID NO: 6) (primer MLV-FORoverlapped RSV-BACK). These two fragments were fused and a productamplified by PCR with the primers RSV-FOR and MLV-BACK. The resultingPCR product which contained a chimeric env gene was cleaved by Kpn I andNot I. The env gene was removed from RCASBP(A)NOT by digesting with KpnI and Not I, and a chimeric Kpn I-Not I fragment was inserted,generating a final plasmid RCAS-M(4070A). The correct sequence of theregion containing the junction between RSV and MLV env sequences wasconfirmed by sequencing using Sequenase Sequencing Kit (USB, OH). Thestructure of RCAS-M(4070A) is shown schematically on FIG. 3A.

Production of Recombinant Viral Particles by RCAS-M(4070A)

To test the expression of a chimeric env gene in a context of an RSVgenome and the ability of the vector to produce particles that hadincorporated the MLV surface glycoproteins, CEFs were transfected withthe RCAS-M(4070A) DNA, as described in Example 1. CEFs transfected withthe pR4070A, the molecular clone of the amphotropic MLV from which theMLV env gene was derived served as a positive control. 24 hr aftertransfection, cell culture medium was harvested and viral particlesrecovered by ultracentrifugation as described in Example 1. As can beseen in FIG. 3B, CEFs transfected with RCAS-M(4070A) generate particlesthat contain the MLV surface glycoprotein (lanes 1 and 2). Lanes 3 and 4of FIG. 3B contain the control viral particles from the cellstransfected with the amphoteric MLV. The incorporation of gp70 into aparticles produced by RCAS-M(4070A) appeared to be approximately asefficient as the incorporation into MLV virions (compare lanes 1 and 2with lanes 3 and 4).

Replication of RCAS-M(4070A) in CEFs

Having demonstrated that the recombinant retroviral vector RCAS-M(4070A)produced chimeric viral particles, its ability to replicate in CEFs wasmeasured. CEF cells were transfected with RCAS-M(4070A) DNA as describedin Example 1 and passaged to allow the virus to spread. A total of sixpassages were done. At passages 1, 4 and 6, a small number of cells wasplated separately, and the ability of the virus to spread was measuredby the staining of CEFs by the antibodies that react with expressedgp70.

The indirect immunofluorescence microscopy assay was performed asfollows. CEFs infected with RCAS-M(4070A) were grown on glass slides.Cells were fixed with methanol, reacted with anti-gp70 goat serum(diluted 1:200 in PBS-l% bovine serum albumin), washed 3 times for 5 minwith PBS containing 0.1% Triton X-100 and incubated with fluoresceinisothiocyanate (FITC)-conjugated rabbit anti-goat secondary antibody.Photomicroscopy was performed by using a Nikon Microphot-FXA microscope(Nicon Inc., Melville, N.Y.). As shown in FIG. 4A, only a small numberof positively stained cells are seen initially (passage 1). This numberincreases significantly by passage 4 and at passage 6 virtually allcells are infected and expressing gp70.

To monitor the production of virus particles, cell culture medium ateach passage was harvested and particles were recovered byultracentrifugation as described in Example 1. The viral particles wereanalyzed by immunoblotting with the antibodies against p19 and gp70 asdescribed in Example 1. The amount of viral proteins in the supernatantincreases by passage 4 and reaches a high level by passage 6 (see FIG.4B). The ability of RCAS-M(4070A) to spread in the CEF culture iscomparable to the rates of spreading seen with the parental vectorRCASBP(A).

The data show that the chimeric retrovirus replicates in the avian cellsat rates comparable to the original RSV-based retroviral vector. Theparticles of RCAS-M(4070A) generated after transfection into CEFscontain approximately the same amount of the gp70 as do particles of theamphotropic MLV, and can infect mammalian cells. Taken together, thesedata suggest that the initial association of the RSV gag and theamphotropic MLV env proteins is efficient enough to allow the assemblyof the infectious virions.

                  TABLE 1                                                         ______________________________________                                        Titer of RCAS-M(4070A)Puro on mammalian cells.                                      Species      Cell line    Titer                                         ______________________________________                                        Mouse          NIH 3T3      7.1 × 10.sup.4                                Dog D17 5-7 × 10.sup.5                                                  Human HeLa 3.3 × 10.sup.4                                             ______________________________________                                    

Gene Transfer to Mammalian Cells by RCAS-M(4070A).

To demonstrate the ability of RCAS-M (4070A) viral particles to infectand transfer genes to mammalian cells, the marker gene pac, that confersresistance to the antibiotic puromycin (de la Luna, et al. (1988) Gene62:121-126, Vara et al., (1986) Nucleic Acids Res. 14:4617-4624), wasintroduced into the Cla I cleavage site downstream of the env gene andsplice acceptor to generate the vector RCAS-MC (4070A) Puro.

To facilitate cloning into RCAS-M(4070A), a silent point mutation wasused to eliminate the Cla I site in the amphotropic env region. It wasintroduced by site-directed mutagenesis. The Kpn I-Not I-fragment ofRCAS-M(4070A) was first subcloned into pBluescript KS II formutagenesis. Mutant clones selected by Cla I cleavage were propagated inEscherichia coli BMH 71-18 mutS. The env region in RCAS-M(4070A) wasreplaced with the mutant Kpn I-Not I fragment, giving rise to a vectorRCAS-MC(4070A).

To construct RCAS-MC(4070A)Puro, the puromycin resistance gene (pac) wasamplified by PCR from the plasmid pSVpac (de la Luna et al., (1988) Gene62:121-126; Vara et al., (1986) Nucleic Acids Res. 14:4617-4624) withthe primers that appended Nco I and Hind III sites to the 5'- and3'-ends of pac respectively. The product was then cloned into theadaptor plasmid Cla12Nco. Hughes et al., (1987) J. Virology61:3004-3012. The pac gene was isolated from the adaptor construct as aCla I-fragment and introduced into RCAS-MC(4070A).

CEFs were transfected with RCAS-MC(4070A)Puro as described in Example 1and passaged 6 times. Virus-containing culture fluid was used to infectfresh cells that were passaged two more times. The resulting virus wastitered on murine cells (NIH 3T3), human (HeLa) cells and D17, dogkidney cells. Titration on mammalian cells was performed as follows.Virus-containing culture fluid was harvested and filtered through a 0.45μm membrane. Host cells were plated in 60-mm plates (5×10⁵ cells perplate) and grown overnight. Cells were infected with serial dilutions ofthe retroviral vector stocks in the presence of polybrene (10 μg/ml) for24 h, trypsinized and plated in a selective medium containing G418 (400μg/ml, GIBCO BRL) or puromycin (2.5 μg/ml, Sigma). Ten days later,colonies that developed from resistant cells were fixed with methanol,stained with Giemsa stain and counted. In some experiments, individualcolonies were isolated and expanded into cell lines for furtheranalysis.

Puromycin-resistant colonies of D17 cells produced by infecting thecells with the serial dilutions of RCAS-MC(4070A)Puro are shown in FIG.5.

Table 1 shows that RCAS-MC (4070A) Puro was able to infect each of themammalian cells tested in high titers. The titer of RCAS-MC(4070A)Puroon D17 (dog) cells was approximately a one order of magnitude higherthan the titer on NIH 3T3 (mouse) or HeLa (human) cells. The variableefficiency with which RCAS-MC(4070A)Puro infects these cells could beattributed to the differences in the amphotropic receptor density on thesurface of mammalian cells of different tissues and species.

Replication and Infectivity of RCAS-M(4070A)NEO

Additionally, the marker gene neo was introduced into RCAS-M(4070A). Theresulting vector, RCAS-M(4070A)NEO (FIG. 6A), was used to infect andtransfer genes to mammalian cells. RCAS-M(4070A)NEO was constructed asfollows: The ClaI-fragment containing the neo gene was isolated fromRCASBP(A)NEO and cloned into the ClaI site of RCASBP(A)NOT. The chimericenv gene was transferred into this plasmid as a KpnI-NotI fragment.

CEFs were transfected with RCAS-M(4070A)NEO as described in Example 1,and viral stocks that were generated 24 hours after transfection weretitered on murine NIH 3T3 and human HeLa cells as described above. Thevirus obtained by the transient expression of the RCAS-M(4070A)NEO inCEFs had the titer 2-3×10³ colony forming units (cfu/ml) on both celllines. Genomic DNA isolated from the G418^(r) clones was analyzed bysouthern blot hybridization. Cells were infected by RCAS-M(4070A)NEO andG418^(r) clones were selected. Their genomic DNA was digested with EcoRIand BamHI. Fragments were resolved in agarose gel, transferred ontocharged nylon membrane, and hybridized with digoxygenin-labeledEcoRI-digested RCAS-M(4070A)NEO DNA. Membranes were incubated withalkaline phosphatase-conjugated anti-digoxygenin antibody, and bandswere detected using Lumi-Phos 530 reagent. As shown in FIGS. 6B and C,the majority of the G418r clones derived from NIH 3T3 and HeLa cellscontained provirus that was structurally indistinguishable from theRCAS-M(4070A)NEO.

The transfected CEFs were passaged and the produced virus was used toinfect both fresh CEFs and mammalian cells. By immunoblotting analysisof the viral particles collected at each passage, it was determined thatthe virus replicated reasonably efficiently in CEFs; however, theability to transfer neo^(r) into NIH 3T3 cells decreased relativelyrapidly (Table 2) and was several orders of magnitude lower than thetiter of RCAS-M(4070A)Puro at a similar passage (compare Table 1 andTable 2). Sequencing of the proviral DNA as described in Example 5showed that the neo gene was somewhat unstable in the context of theamphotropic env gene and specifically that a number of provirusessuffered deletions involving the splice acceptor site, initiation codonand 5' end of the neo gene.

It appears that the difference in stability between the neo and pacselectable markers is not a function of the vector, but rather a measureof the relative cytotoxicity of these markers on the host cell. It seemsunlikely that there is a much greater tendency for the neo insert torearrange because this would imply that there are certain preferredsites for rearrangement in the neo insert. However, the neo deletionsthat were analyzed were all different.

                  TABLE 2                                                         ______________________________________                                        Titer of RCAS-M(4070A)NEO on NIH 3T3 cells                                           Virus         Titer, cfu/ml                                            ______________________________________                                        24 h posttransfection                                                                          2.6 × 10.sup.3                                           Virus passage 1 10.sup.1                                                      Virus passage 2 10.sup.1                                                      Virus passage 3   5 × 10.sup.2                                        ______________________________________                                    

EXAMPLE 3 Transmembrane Protein p15E is Correctly Processed inRCAS-M(4070A) Particles

In the murine leukemia viruses, the env precursor is initially cleavedby a cellular protease into gp70 (SU) and pre-p15E (TM). After the virusparticle is released from the cell, the viral protease removes theC-terminal 16 residues from the cytoplasmic domain of pre-p15E, yieldingthe mature p15E and p2E. Rein et al., (1994) J. Virology 68:1773-1781.This cleavage activates the membrane fusion capability of the envprotein and is essential for viral infectivity.

Virions produced by cells infected with RCAS-M(4070A) do not contain MLVprotease. However, the chimeric virus is infectious, suggesting thatpre-p15E is cleaved. Viral particles were prepared as described inExample 1 and the proteins were fractionated on 16% SDS-PAGE andanalyzed by immunoblotting with rabbit antiserum against p15E (kind giftof Alan Rein) (FIG. 7). The data shows that the chimeric virus particlesformed by RCAS-M(4070A) contain the processed p15E, although the MLVprotease is not present. The transmembrane protein processing in theRCAS-M(4070A) particles appears to be as efficient as in the wild typeMLV virions (compare lanes 1 "MLV" and 3 "RCAS-M (4070A)" of FIG. 7),suggesting that either RSV protease or the protease of the avian cellscan effectively process pre-p15E.

EXAMPLE 4 Virus Particles are not Produced by Mammalian Cells Infectedwith RCAS-M(4070A)

Recent studies demonstrated the development of the lymphomas in thenon-human primates after gene transfer as a result of infection andspreading of the replication-competent helper MLV which was present inthe retroviral vector stocks. (Donahue et al., (1992) J. Exp. Med.176:1125-1135; Vanin et al., (1994) J. Virology 68:4241-4250). Thisillustrates the considerable importance of the issue of a retroviralvector safety. One of the advantages of using avian retrovirus-basedvectors in a mammalian cell system is that the mammalian cellstransfected or infected with RSV do not produce infectious viralparticles (Federspiel et al., (1994) PNAS (U.S.A.) 91:1124-11245) andtherefore the vector cannot spread in the mammalian host. Inability ofRSV to synthesize or to export into the cytoplasm a full-length genomicRNA, (Berberich et al., (1990) J. Virology 64:4313-4320; Knight et al.,(1993) J. Gen Virology 74(Pl12):2629-2636; Quintrell et al., (1980) J.Mol. Biol. 143:363-393), as well as its inability to process gagprecursor protein in mammalian cells (Vogt et al., (1982) J. Virology44:725-730) were implied as the possible defects that prevent thesynthesis of the virus structural proteins and production of the virusparticles.

To determine whether the mammalian cells infected by RCAS-M(4070A)generate virus particles, we infected fresh NIH 3T3 cells with thesupernatants of several NIH 3T3/RCAS-M(4070A)NEO clones carryingun-rearranged proviruses. Cells were then subjected to G418 selection.No G418-resistant colonies were detected. This sensitive assay showedthat infectious viral particles were not produced by the murine NIH 3T3cells containing non-rearranged RCAS-M(4070A)NEO provirus. Additionally,this experiment was repeated using RCAS-M2 (4070A)Puro to infect NIH 3T3cells. Again, no viral particles were detected. Thus, the RSV-basedvector RCAS-M(4070A), being inherently replication defective in themammalian cells, has substantially improved safety features required forsensitive applications such as human gene therapy.

EXAMPLE 5 Construction of RCAS-M2(4070A), A Fast Replicating and StableVector

Initially the chimeric virus RCAS-M(4070A) replicates at a considerablylower rate than the parental vector RCASBP(A). Indeed, aftertransfection of RCAS-M(4070A) into CEFs, 3-4 cell passages were requiredbefore detectable amounts of the virus were produced. However, after 5-6passages on CEFs, the chimeric virus infects these cells efficiently andspreads quickly throughout the entire culture.

To investigate the possibility that genetic changes in the viral genomeduring this period of initial slow replication permitted the chimericvirus to adapt and grow more efficiently, the env regions of molecularclones of RCAS-M(4070A) provirus containing the neo marker gene(RCAS-M(4070A)NEO) were sequenced.

Cloning of RCAS-M(4070A)NEO Provirus

RCAS-M(4070A)NEO provirus was cloned as follows. Low-molecular-weightDNA was isolated from CEFs infected with RCAS-M(4070A)NEO at passage 3by the Hirt extraction procedure, Hirt (1967) J. Mol. Bio. 26:365-369.The low-molecular-weight DNA was cleaved with the restrictionendonuclease SacI, ligated with SacI-cleaved vector λZAP Express(Stratagene, Calif.) and packaged using GigaPack II Gold packagingextract (Stratagene, Calif.). A library of 4.7×10⁶ independent cloneswas screened by hybridization with a ³² P-labeled amphotropicenv-specific probe (SalI/ClaI fragment derived from pR4070A). Positivephage clones were converted into plasmids by co-infection with ExAssistHelper phage (Stratagene, Calif.). These clones were designated env1,env3, env6, env8, and env9. RCAS-M(4070A)NEO proviral DNA in theresulting plasmid clones was analyzed by cleavage with restrictionendonucleases.

Sequencing of the Env Regions of the RCAS-M(4070A)NEO Proviruses

The env gene sequences of the RCAS-M(4070A)NEO provirus clones and theparental plasmid RCAS-M(4070A)NEO were determined by cycle sequencing ofboth strands using PRISM™ Ready Reaction DyeDeoxy Terminator CycleSequencing Kit (Applied Biosystems, CA). Sequencing reactions wereanalyzed using the Automated 373A DNA Sequencer (Applied Biosystems,CA). The complete sequences of the env genes were assembled usingSequencher software (Gene Codes Corporation, MI).

A limited spectrum of mutations in the env region of the individualproviral clones was discovered. The mutations were located primarily inthe central and C-terminal regions of the gp70 coding sequence (FIG. 8).In all of the clones, two adjacent cytosines in the region encoding gp70were mutated to A and T, resulting in the substitution of Ile for Pro atposition 242. The same region of the env gene in the parental plasmidRCAS-M(4070A)NEO, had a proline codon at position 242, thus confirmingthat the Pro242Ile mutation had been selected during the passage of thevector on CEFS, and had not occurred in the course of the vectorconstruction. In addition to this mutation, other substitutions werefound, including Lys308Gln (clones env1 and env9), Ala310Gly (clonesenv3 and env6), and Met446Leu (clones env6 and env9). Some clones hadadditional amino acid changes, which were seen only in that clone. Inaddition, in the clone env3, the sequence encoding the C-terminal end ofthe gp70 and almost all of the p15E was deleted.

The Pro242Ile mutation appears to be critically important for efficientreplication, since it is present in all of the clones we derived frompassaged RCAS-M(4070A)NEO virus. Furthermore, this mutation alone issufficient to dramatically increase the efficiency of the virusreplication, since it is the only mutation found in the env gene ofclone env8, the virus that replicates most efficiently in CEFs andproduces the highest titers on mammalian cells.

The replacement of Pro242 with the uncharged isoleucine residue couldindicate an important structural change in the gp70, since prolineresidues are often located at the points of β-turns. This change couldreflect the adjustment necessary for the gp70 to recognize both avianand mammalian types of the amphotropic receptor or, alternatively, couldenhance the interaction between RSV capsid and the env protein from theamphotropic MLV. It is also possible that the mutation affects thestructure and/or function of viral RNA.

The mammalian cellular receptor for amphotropic MLV has recently beencloned and characterized. Miller, et al., (1994) PNAS (USA) 91:78-82;van Zeijl et al., (1994) PNAS (USA) 91:1168-1172. However, the gene forthe avian amphotropic receptor has not been unambiguously identified.van Zeijl et al., (1994) PNAS (USA) 91:1168-1172. Passaging ofRCAS-M(4070A) through CEFs could select for the viral variants that haveincreased affinity for the avian amphotropic receptor. No mutations wereseen either in the N-terminal half of gp70, the region that appears todetermine host range (Ott and Rein (1990) J. Virol 64:757-766; Ott andRein (1992) J. Virol 66:4632-4638) or in the hypervariable proline-richregion. If the alteration in gp70 increases the affinity of the proteinfor the avian receptor, it does not compromise the ability of the virusto infect mammalian cells.

Construction of RCAS-M2(4070A)

To construct an "adapted" version of RCAS-M(4070A) which takes advantageof the genetic changes which occurred in the viral genome during theperiod of initial slow replication, vectors designatedRCAS-M2(4070A)Puro1, 6, 8 and 9 were constructed. These vectors containthe (gag-pol)^(RSV) -env^(MLV) regions derived from clones env 1, env 6,env 8 and env 9 of RCAS-M(4070A)NEO in place of the (gag-pol)^(RSV)-env^(MLV) region of RCAS-M(4070A)Puro.

To construct RCAS-M2(4070A)Puro, DNA fragments containing the(gag-pol)^(RSV) -env^(MLV) region were derived from the plasmid clonesby digestion with SacI and NotI and ligated with the LTR-Puro-LTRcassette that was obtained by SacI/NotI cleavage of RCAS-MC(4070A)Puro.

The resulting RCAS-M2(4070A)Puro1, 6, 8, and 9 vectors were transfectedinto CEFs, and the cells were passaged to generate virus. At eachpassage, the production of viral particles was quantified by determiningthe virion-associated RT activity, and the number of viruses able toinfect mammalian cells was determined by titration on D17 cells asdescribed in Example 2.

Determination of the virion-associated RT activity was performed asdescribed in Whitcomb et al., (1995) J. Virol 69:6228-6238. Briefly,virus particles were recovered from the culture fluid by centrifugationat 14,000 rpm for 30 min at 4° C. Pellets were resuspended in RT buffer(50 mM Tris, pH 8.3, 60 mM NaCl, 12 mM MgCl₂, 20 mM dithiothreitol, 0.1%NP-40) containing oligo(dG) (5 μg/ml), poly(rc) (10 μg/ml), dGTP (10 μM)and 10 μCi of [α-³² P]dGTP (800 Ci/mmol, Amersham, Ill.). Reactionmixtures were incubated at 37° C. for 1 h. Nucleic acids wereprecipitated by adding 1 ml of 10% trichloracetic acid (TCA), collectedon the GF/C glass microfiber filters (Whatman, Oreg.) and washed with10% TCA and 95% ethanol. Filters were dried and counted in a TriCarb1500 Liquid Scintillation analyzer (Packard).

As shown in FIG. 9A, the initial rates of replication of the "adapted"vectors are much faster than the parental vector, RCAS-M(4070A)Puro.Virus production is detectable at passage 1 and reaches a maximum atpassages 3-4, which is similar to the initial rates of replication ofRCASBP(A). In this experiment, the production of the parental vector,RCAS-M(4070A)Puro, designated "env (wt)," could not be detected untilpassage 6. The "adapted" RCAS-M2(4070A)Puro vectors are infectious formammalian cells, exhibiting significant titers on D17 cells (FIG. 9B).The fastest replicating virus (clone env8) reaches the maximum titer onD17 cells by passage 2 (compare FIGS. 9A and B).

Construction of RCAS-M2C(4070A)

To facilitate cloning into RCAS-M2(4070A), the ClaI site in the env geneof RCAS-M2(4070A)Puro (derived from the clone env8) was eliminated byintroducing a silent point mutation by site-directed Mutagenesis asdescribed in the construction of RCAS-MC(4070A) in Example 2. The DNA ofthe resulting vector RCAS-M2C(4070A)Puro was transfected into CEFs asdescribed in Example 1 and the cells were passaged. At each passage, thenumber of virus particles able to infect mammalian cells was quantifiedby titration on D17 cells as described in Example 2. As can be seen inFIG. 9C, the RCAS-M2C(4070A)Puro replicates efficiently in CEFs, and thetiter of a virus stock produced at each passage is the same as the titerof the RCAS-M2(4070A)Puro virus from which it was derived.

In clinical applications of retroviral gene transfer, both the titer andthe safety of the vector are of critical importance. The titer of theRCAS-M2C(4070A)Puro vector exceeds 10⁶ cfu/ml. This titer can beobtained by 2-3 cell passages after transfection of CEFs. Not only isthe vector simple to use and the titer high, but the vector should alsobe quite safe. RSV-infected mammalian cells do not produce infectiousviral particles and the vector cannot spread in the mammalian host. Theinability of RSV to efficiently export a full-length genomic RNA intothe cytoplasm (Berberich et al., (1990) J. Virol 64:4313-4320; Knight etal., (1993) J. Gen Virol 74(Pt 12):2629-2636; Nasioulas et al., (1995)PNAS (USA) in press; Quintrell et al., (1980) J. Mol. Biol. 143:363-393)as well as its inability to assemble, export, and process the Gagprecursor protein in mammalian cells (Nasioulas et al., (1995) PNAS(USA) in press, Vogt et al., (1988) J. Virol 44:725-730) appear toprevent the production of infectious virus particles. To determinewhether the mammalian cells infected by RCAS-M(4070A)NEO generateinfectious virus particles, we infected fresh NIH 3T3 cells with thesupernatants of several NIH 3T3/RCAS-M(4070A)NEO clones carryingunrearranged proviruses as described in Example 4. Following G418selection, no G418-resistant colonies were detected, indicating that themammalian cells infected with RCAS-M(4070A) do not produce infectiousparticles. No infectious viral particles were detected in the murine NIH3T3 cells containing non-rearranged RCAS-M(4070A)NEO provirus weredetected using G418 selection as described in Example 4. Similarexperiments were performed using RCAS-M2(4070A)Puro to infect themammalian cells and no virus particles were detected in the infectedmurine NIH 3T3 cells. Since it is inherently defective in the mammaliancells, the RSV-based vector RCAS-M2C(4070A) appears to have the safetyfeatures required for such sensitive applications as human gene therapy.This vector, designated RCAS-M2C (4070A) or RCASBP-M2C (4070A) in thisapplication, was deposited with the American Type Culture Collection,12301 Parklawn Drive, Rockville, Md. 20852 on Sep. 23, 1998 under ATCCaccession number 203326.

For purposes of clarity of understanding, the present invention has beendescribed in some detail by the use of illustration and examples.However, it will be obvious to those skilled in the art that certainmodifications may be practiced within the scope of the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 6                                        - - <210> SEQ ID NO 1                                                        <211> LENGTH: 30                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Unknown Organism                                              <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Unknown Or - #ganism: NUCLEIC       ACIDS                                                                                 PROBES                                                                   - - <400> SEQUENCE: 1                                                         - - aaaagagctc ggccgacacc cagagtggac         - #                  - #               30                                                                     - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 33                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Unknown Organism                                              <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Unknown Or - #ganism: NUCLEIC        ACIDS                                                                                 PROBES                                                                   - - <400> SEQUENCE: 2                                                         - - aaaagagctc tcatggctcg tactctatgg gtt       - #                  -      #         33                                                                     - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Unknown Organism                                              <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Unknown Or - #ganism: NUCLEIC       ACIDS                                                                                 PROBES                                                                   - - <400> SEQUENCE: 3                                                         - - ggacgaggtt atgccgctgt g           - #                  - #                      - #21                                                                  - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 46                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Unknown Organism                                              <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Unknown Or - #ganism: NUCLEIC        ACIDS                                                                                 PROBES                                                                   - - <400> SEQUENCE: 4                                                         - - acattaaaga cctgatgggg gctaacatca gctcttaccc ccgtaa   - #                     46                                                                        - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Unknown Organism                                              <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Unknown Or - #ganism: NUCLEIC        ACIDS                                                                                 PROBES                                                                   - - <400> SEQUENCE: 5                                                         - - agcccccatc aggtctttaa tgt           - #                  - #                    23                                                                     - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 32                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Unknown Organism                                              <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Unknown Or - #ganism: NUCLEIC        ACIDS                                                                                 PROBES                                                                   - - <400> SEQUENCE: 6                                                         - - agcggccgct catggctcgt actctatggg tt       - #                  - #              32                                                                   __________________________________________________________________________

We claim:
 1. An isolated nucleic acid molecule comprising:a. at leastone long terminal repeat (LTR) isolated from an avian sarcoma leukosisvirus (ASLV): b. a first nucleic acid sequence selected from a gagregion isolated from ASLV: c. a second nucleic acid sequence selectedfrom a pol region isolated from an ASLV: and d. a third nucleic acidsequence selected from a viral envelope region, said third sequenceencoding a protein which binds to both avian and mammalian cells wherebya viral particle harboring said nucleic acid molecule can infect bothavian and mammalian cells in titers of at least 10⁴ /ml, wherein saidviral particle is replication competent in avian cells but isreplication defective in mammalian cells.
 2. The nucleic acid moleculeof claim 1 further comprising at least a fourth nucleic acid sequence.3. The nucleic acid molecule of claim 1 further comprising a restrictionendonuclease site allowing for the insertion of a further nucleic acidsequence.
 4. The nucleic acid molecule of claim 1 wherein the LTR, thegag and the pol sequences are isolated from Rous sarcoma virus (RSV). 5.The nucleic acid molecule of claim 1 wherein the envelope sequence isisolated from the amphotropic murine leukemia virus (MLV).
 6. Thenucleic acid molecule of claim 1 wherein the envelope sequence is achimeric envelope sequence comprising a sequence encoding the N terminalsignal peptide of an ASLV env region and a sequence selected from thecoding region of an amphotropic MLV env sequence.
 7. The nucleic acidmolecule of claim 5 or 6 wherein the envelope sequence contains anisoleucine at position
 242. 8. The nucleic acid molecule of claim 7wherein the envelope sequence is adapted for increased initialreplication.
 9. The nucleic acid molecule of claim 1 wherein saidnucleic acid molecule comprises RCAS-M (4070A) shown in FIG.
 3. 10. Thenucleic acid molecule of claim 1 wherein said nucleic acid moleculecomprises RCAS-M2C(4070A)Puro
 8. 11. The nucleic acid molecule of claim1 wherein said nucleic acid comprises RCAS-M2C(4070A), deposited as ATCCaccession no.
 203326. 12. A viral particle comprising the nucleic acidmolecule of claim
 1. 13. A viral particle comprising the nucleic acidmolecule of claim
 10. 14. A method for inserting at least one nucleicacid sequence of interest into a host cell in vitro comprisingintroducing the nucleic acid molecule of claim 2 or 10 into the hostcell.
 15. A method for inserting at least one nucleic acid sequence ofinterest into a host cell in vitro comprising infecting the host cellwith the viral particle of claim 12, wherein the infection of said hostcell with said viral particle results in the insertion of a nucleic acidmolecule comprised by said viral particle in the host cell.
 16. A methodfor inserting at least one nucleic acid sequence of interest into a hostcell in vitro comprising infecting the host cell with the viral particleof claim 13, wherein the infection of said host cell with said viralparticle results in the insertion of a nucleic acid molecule comprisedby said viral particle in the host cell.
 17. An isolated host cellcontaining the nucleic acid molecule of claim
 1. 18. An isolated hostcell containing the nucleic acid molecule of claim 10.